Project Summary/Abstract Our team has developed novel approaches for the generation of bladder cancer models based on human patient-derived bladder organoids and xenografts, as well as genetically-engineered mouse models (GEMMs). In particular, we have developed an innovative methodology for three-dimensional culture of organoids that recapitulate the histopathological and molecular properties of their corresponding parental tumors, and have mutational profiles characteristic of human bladder cancer. In addition, we have pioneered the development of GEMMs of muscle-invasive bladder cancer (MIBC), and have demonstrated their utility for co-clinical investigations. Together, these resources provide the foundation for the generation and analysis of a range of in vitro and in vivo models of urothelial cancer, which will serve the needs of this Program Project as well as the broader community of bladder cancer researchers. The Bladder Cancer Models Core will support the scientific objectives of our Program Project by generating human and mouse bladder cancer models that will be vital for all three Projects. The Core is structured around two specific aims: In Aim 1, we will establish a biobank of patient-derived bladder cancer organoid lines, including from patients with rare bladder cancer subtypes and genomic alterations of particular interest to the Program Project, and from women and minority patients. In collaboration with Core A, we will perform histopathological and molecular analyses to assess the similarity of the organoids to their corresponding parental tumors, and will use targeted exome sequencing to categorize their mutational profiles. Our goal is to generate a biobank of organoid lines that is representative of the full spectrum of bladder cancer as well as of a diverse patient population. These organoid lines will be utilized by all three Projects, but will be particularly important for Project 3, which will investigate tumor heterogeneity and clonal evolution in patient- derived organoids. In Aim 2, we will generate and characterize a series of GEMMs of bladder cancer, including those of particular relevance for the Program Project, namely Kdm6a, Arid1a, and Kmt2d, which encode epigenetic regulators that are frequently mutated in human bladder cancer, and Ercc2, a nucleotide excision repair pathway gene that is associated with cisplatin response. Using conditional alleles that have been obtained for each of these genes, we will generate GEMMs based on their loss-of-function alone or in combination with Trp53flox/flox; Ptenflox/flox mice, which represents a well-characterized GEMMs of MIBC. Together with Core A, we will perform histopathological and molecular analyses to assess the relationship of these GEMMs to human bladder cancer. These GEMMs will be important for Projects 1 and 2, which will investigate the functions of epigenetic regulators in bladder cancer, and for Project 3, to complement studies in human organoids. Finally, our work will be of considerable value beyond this Program Project by providing clinically-relevant models for the broader community that will facilitate development of new treatments.